The invention concerns a method for fixing and embedding tissues for histological preparations.
Two types of methods are currently used in this field, namely
1. slicing of frozen sections
2. slicing of fixed and embedded tissue sections.
Slicing of Frozen Sections
Freezing fulfils the dual function of fixing the tissues in the condition in which they are found and to solidify them to enable them to be sliced.
Slicing of frozen samples provides a histological material:
which exhibits substantially no molecular denaturation and thus enables good determination with respect to the nature of the molecules present
which has the disadvantage that it appears in the form of thick sections, thus not enabling a microscopic examination at great magnification, and in which the morphological structure of the tissue has been greatly altered by the freezing
such that, if it is possible to label the molecules, it is not possible to reliably locate the molecules thus labelled in the morphological structure of the tissue.
Finally, the slices made in the frozen tissue are then prepared and examined at room temperature, and thus a chemical fixing is still necessary after the cut.
Slicing of Fixed and Embedded Tissue Sections
In this method, the tissue is (1) first fixed, then (2) subjected to a dehydration step and finally (3) subjected to an embedding step to solidify the tissue and enable it to be cut, after which (4) the tissue is cut into fine lamellae, generally by a microtome. These lamellae are disposed on object slides and (5) these preparations are finally rid of the embedding product and rehydrated prior to the histochemical treatment.
The purpose of the fixing step (1) is to prevent substantially any physicochemical alteration of the tissue, to maintain it in the initial state in which it was collected and to thus permit determinations, in particular, immunological ones.
The purpose of the infiltration/embedding step (3) is to solidify the tissue to make it possible to make the slices as thin as possible, thus enabling a more extensive examination.
The dehydration step (2) is an intermediate step whose purpose is to replace the water present in the tissues by a compound which is itself substitutedxe2x80x94possibly with the use of solventsxe2x80x94by the infiltration/embedding compound.
Step (4) provides the embedded, and thus solidified, tissue sections and step (5) finally restores the tissue more or less to its initial condition, permitting the desired analysis.
Various liquid fixatives are known for implementing the fixing step (1), such as compositions based on soluble zinc salts, various organic compounds (see e.g. EP-A-0562877), as well as acetone (see e.g. U.S. Pat. No. 5,104,640).
Various compounds, such as e.g. ethanol, methanol, isopropanol and acetone are known for the dehydration step (2) (see e.g. the article by Beckstead cited below).
For the infiltration/embedding step (3), paraffin is conventionally currently used. The disadvantage of the use of paraffin is that it is weakly soluble and consequently it is only possible to eliminate it by resorting to strong solvents and that it only melts at temperatures of 58-60xc2x0 C. This is substantially higher than the physiological temperature of tissues and thus denaturing for them.
Jay H. Beckstead, in The Journal of Histochemistry and Cryochemistry (xe2x80x9cA Simple Technique for Preservation of Fixation-sensitive Antigens in Paraffin-embedded Tissuesxe2x80x9d, Vol. 42, No. 8, pp. 1127-1134, 1994) describes a method for fixing/embedding using a zinc salt fixative, together with a paraffin embedding, and comparing it, on the one hand, with known paraffin embedding methods together with other fixatives and, on the other hand, with the freezing method.
This method consists of
fixing the tissues with soluble zinc salts in a buffer solution,
dehydrating the tissues thus fixed by increasing concentrations of ethanol/isopropanol,
eliminating the ethanol/isopropanol with xylene,
infiltrating/embedding with paraffin at 58-60xc2x0 C.,
making the slices,
eliminating the paraffin with three xylene baths, followed by three isopropanol baths, and rehydrating.
Acetonexe2x80x94which also acts as a dehydrating agentxe2x80x94is known for causing a perceptible morphological denaturation of the tissues. For this reason, researchers consider it to be inappropriate for fixing when one wishes to make morphological determinations, which is quite generally the case for histological sections prepared for examination with a microscope.
Alcohols (ethanol, methanol, isopropanol, for example) are known for not perceptibly degrading morphological structures when they are used with care. They are, therefore, conventionally used for the dehydration of tissues. On the other hand, they have a marked degradation effect on molecular structures (by coagulation) and, consequently, they have disadvantages when both the molecular and the morphological structures are to be preserved, as is the case in immunological studies in situ and ex vivo in immunohistochemistry.
On the other hand, alcohols have a very great dehydrating power and, to avoid altering the morphological structure, it is necessary to carry out the dehydration by successive steps, with increasing alcohol concentrations, which obviously hampers the procedure.
Having regard to the fact that paraffin is generally of low solubility, it is necessary to provide, between the dehydration step and the embedding step, an intermediate step to eliminate the dehydrating agent with solvents such as toluene, xylene, benzene or the like, which creates denaturation problems for tissues, and toxicity, and adds still another step to the mode of operation.
Finally, impregnation with paraffin requires temperatures of xc2x160xc2x0 C., higher than the physiological temperature, and this exerts a denaturing effect (thermal coagulation) on the molecular structures.
Moreover, the process for eliminating paraffin and rehydration of the preparations must also be done with strong solvents which are not without disadvantages.
A method is also known (xe2x80x9cImmunofluorescence Detection of F-actin on Low Melting Point Wax Sections from Plant Tissuesxe2x80x9d by Stanislav Vitha et al, in The Journal of Histochemistry and Cryochemistry, Vol. 45(1): 09-95, 1997) that consists of
fixing the tissues successively in formaldehyde and zinc chloride solutions, the latter step being carried out at 60xc2x0 C.,
dehydrating the tissues with increasing ethanol concentrations,
infiltrating/embedding, in several steps, the tissues in a resin consisting of 90% polyethylene glycol 400 distearate [which is also known under the name polyoxyethylene bis(stearate)] and of 10% 1-hexadecanol, at 35-57xc2x0,
making the slices, and
eliminating the resin with ethanol of technical grade, and rehydrating the preparations.
According to the invention, a method for fixing/embedding is to be provided which does not perceptibly denature either the molecular structure nor the morphological structure of the tissues, to enable analysis that is as reliable and precise as possible. This is important, in particular, for immunological analysis in situ and ex vivo, in which determinations and immunological analyses are to be made not only on biological molecules extracted from their cellular and/or tissue environment, but also such molecules in this environment.